Does the restriction endonuclease EcoRV employ a two-metal-Ion mechanism for DNA cleavage?
Two models for the catalytic mechanism of the restriction endonuclease EcoRV exist which differ in the number and function of metal ions proposed to be directly involved in catalysis. In one model, two metal ions bound by Glu45, Asp74, and Asp90 are assumed to have a direct catalytic function; in the other, only one metal ion bound by Asp74 and Asp90. We show here that in the presence of Mn2+, the catalytic activity of an EcoRV-E45A mutant is only slightly reduced (1.8-fold) as compared to wild type EcoRV and that the single-turnover rate constant of DNA cleavage by E45A is reduced only 39-fold, whereas the D74A and D90A mutants are catalytically inactive under all conditions. These findings make an important catalytic function of Glu45, like binding of an essential divalent metal ion, unlikely. In addition, we have analyzed the dependence of the DNA cleavage rate by EcoRV and EcoRV mutants on the concentration of Mg2+ and Mn2+. We found for the wild type enzyme a sigmoidal dependence
